Fgfr1 agonists and methods of use

ABSTRACT

The invention provides nucleic acid molecules encoding anti-fibroblast growth factor receptor-1 (FGFR1) antibodies and vectors and host cells comprising the nucleic acid molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/754,229, filed Jun. 29, 2015 which is a divisional of U.S.application Ser. No. 13/472,352, filed May 15, 2012, now U.S. Pat. No.9,085,626, which claims benefit of priority to U.S. Patent Applications61/486,731, filed May 16, 2011, and 61/536,936, filed Sep. 20, 2011, theentire contents of each of which are incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 31, 2017, isnamed Sequence Listing.txt and is 26,144 bytes in size.

FIELD OF THE INVENTION

The present invention relates to FGFR1 agonists and methods of using thesame.

BACKGROUND

The inability to control blood glucose levels underlies a variety ofmetabolic conditions. Diabetes is a hyperglycemic syndrome that resultsfrom a defect in insulin secretion in response to glucose (type 1 andtype 2 diabetes) and decreased insulin effectiveness in stimulatingskeletal muscle glucose uptake and in restraining hepatic glucoseproduction (type 2 diabetes). Diabetes it a highly prevalent diseaseand, although therapeutic options are available for some diabetics,there is an urgent need for additional therapies.

Fibroblast growth factor 21 (FGF21) is a member of the endocrine FGFsubfamily, that includes FGF19 and FGF23, and it has been identified asa potential disease-modifying agent to reverse obesity andobesity-induced hepatosteatosis and hyperglycemia (see, e.g.,Kharitonenkov and Larsen, Trends Endocrinol. Metab. 22(3):81-6 (2011);Kharitonenkov et al., J. Clin. Invest. 115: 1627-35 (2005); WO2010/042747). The endocrine FGF21 protein binds to three FGF receptors(FGFRs 1-3), and improves insulin resistance and type 2 diabetes bythese receptors together with their membrane bound co-receptorbeta-Klotho. However, its development has been hampered by its poorpharmacokinetics and poor understanding of the biological mechanism ofaction. Anti-FGFR1 antagonist antibodies have also been proposed for thetreatment of diabetes (WO 2005/037235). However, the identity of whichof the three FGFRs mediates the beneficial metabolic activity of FGF21has not been discovered.

SUMMARY

The invention is based, in part, on the discovery that activation ofFGFR1 ameliorates diabetes. The invention provides FGFR1 agonists,including agonist anti-FGFR1 antibodies and methods of using the same.

In one aspect, the invention provides a method of treating a metabolicdisease or condition in an individual, comprising administering to theindividual an effective amount of an anti-fibroblast growth factorreceptor-1 (FGFR1) agonist, wherein the metabolic disease is selectedfrom the group consisting of: polycystic ovary syndrome (PCOS),metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia,hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes,latent autoimmune diabetes (LAD), and maturity onset diabetes fo theyoung (MODY). In some embodiments, the FGFR1 agonist does not activateFGFR2 or FGFR3. In some embodiments, the FGFR1 agonist is an anti-FGFR1antibody. In some embodiments, the anti-FGFR1 antibody has twoFGFR1-binding sites, e.g. a full-length antibody or a F(ab′)2 fragment.In some embodiments, the antibody binds to peptide #26 KLHAVPAAKTVKFKCP(SEQ ID NO: 28) or peptide #28 FKPDHRIGGYKVRY (SEQ ID NO: 29). In someembodiments, the antibody binds to both peptide #26 and peptide #28. Insome embodiments, the anti-FGFR1 antibody binds to both FGFR1b andFGFR1c. In some embodiments, the antibody is a bispecific antibody. Insome embodiments, the bispecific antibody also binds to beta-Klotho.

In another aspect, the invention provides an isolated antibody thatbinds to FGFR1, wherein the antibody is an agonist of FGFR1 activity. Insome embodiments, the antibody is not an agonist of FGFR2 or FGFR3. Insome embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is a human, humanized, or chimeric antibody.In some embodiments, the antibody comprises (a) HVR-H3 comprising anamino acid sequence selected from the group consisting of SSGYGGSDYAMDY(SEQ ID NO: 16), SGYGGSDYAMDY (SEQ ID NO: 17), EHFDAWVHYYVMDY (SEQ IDNO: 18), TGTDVMDY (SEQ ID NO: 19), and GTDVMDY (SEQ ID NO: 20), (b)HVR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO: 23), and(c) HVR-H2 comprising an amino acid sequence selected from the groupconsisting of X₁X₂IX₃PX₄DGX₅TX₆YADSVKG, wherein X₁ is A or G, X₂ is D orE, X₃ is D or Y, X₄ is N or Y, X₅ is A or D, and X₆ is D or Y (SEQ IDNO: 24) and X₁IX₂PX₃DGX₄TX₅YADSVKG, wherein X₁ is D or E, X₂ is D or Y,X₃ is N or Y, X₄ is A or D, and X₅ is D or Y (SEQ ID NO: 25). In someembodiments, the antibody comprises (a) HVR-H1 comprising the amino acidsequence GFTFX₁X₂X₃X₄IX₅, wherein X₁ is S or T, X₂ is N or S, X₃ is N orT, X₄ is W or Y, X₅ is H or S (SEQ ID NO: 26), (b) HVR-H2 comprising anamino acid sequence selected from the group consisting ofX₁X₂IX₃PX₄DGX₅TX₆YADSVKG, wherein X₁ is A or G, X₂ is D or E, X₃ is D orY, X₄ is N or Y, X₅ is A or D, and X₆ is D or Y (SEQ ID NO: 24) andX₁IX₂PX₃DGX₄TX₅YADSVKG, wherein X₁ is D or E, X₂ is D or Y, X₃ is N orY, X₄ is A or D, and X₅ is D or Y (SEQ ID NO: 25), and (c) HVR-H3comprising an amino acid sequence selected from the group consisting ofSSGYGGSDYAMDY (SEQ ID NO: 16), SGYGGSDYAMDY (SEQ ID NO: 17),EHFDAWVHYYVMDY (SEQ ID NO: 18), TGTDVMDY (SEQ ID NO: 19), and GTDVMDY(SEQ ID NO: 20). In some embodiments, the antibody comprises (a) HVR-H1comprising the amino acid sequence GFTFTSTWIS (SEQ ID NO: 7), (b) HVR-H2comprising an amino acid sequence selected from the group consisting ofGEIDPYDGDTYYADSVKG (SEQ ID NO: 10) and EIDPYDGDTYYADSVKG (SEQ ID NO:11), and (c) HVR-H3 comprising an amino acid sequence selected from thegroup consisting of SSGYGGSDYAMDY (SEQ ID NO: 16) and SGYGGSDYAMDY (SEQID NO: 17). In some embodiments, the antibody comprises (a) HVR-H1comprising the amino acid sequence GFTFSNNYIH (SEQ ID NO: 8), (b) HVR-H2comprising an amino acid sequence selected from the group consisting ofADIYPNDGDTDYADSVKG (SEQ ID NO: 12) and DIYPNDGDTDYADSVKG (SEQ ID NO:13), and (c) HVR-H3 comprising the amino acid sequence EHFDAWVHYYVMDY(SEQ ID NO: 18). In some embodiments, the antibody comprises (a) HVR-H1comprising the amino acid sequence GFTFTSNWIS (SEQ ID NO: 9), (b) HVR-H2comprising an amino acid sequence selected from the group consisting ofAEIDPYDGATDYADSVKG (SEQ ID NO: 14) and EIDPYDGATDYADSVKG (SEQ ID NO:15), and (c) HVR-H3 comprising an amino acid sequence selected from thegroup consisting of TGTDVMDY (SEQ ID NO: 19) and GTDVMDY (SEQ ID NO:20). In some embodiments, the antibody further comprises (a) HVR-L1comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 21); (b)HVR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO: 22); and(c) HVR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO: 23).In some embodiments, the antibody comprises a VH sequence selected fromthe group consisting of SEQ ID NO: 2, 3 and 4. In some embodiments, theantibody comprises a VL sequence of SEQ ID NO:

6.

In some embodiments, the anti-FGFR1 antibody has two FGFR1-bindingsites, e.g. a full-length antibody or a F(ab′)2 fragment. In someembodiments, the antibody of the invention is a multispecific antibody.In some embodiments, the antibody also binds to beta-Klotho. In someembodiments, the antibody is an IgG1 antibody. In some embodiments, theinvention provides an isolated nucleic acid encoding an antibody of theinvention. In some embodiments, the invention provides a host cellcomprising the nucleic acid of claim 21. In some embodiments, theinvention provides method of producing an antibody comprising culturingthe host cell of claim 22 so that the antibody is produced. In someembodiments, the method further comprises recovering the antibody fromthe host cell.

In some embodiments, the invention provides a pharmaceutical formulationcomprising an antibody of the invention and a pharmaceuticallyacceptable carrier.

In some embodiments, the invention provides an antibody of the inventionfor use as a medicament. In some embodiments, the antibody of theinvention is for use in treating a metabolic disease or conditionselected from the group consisting of: polycystic ovary syndrome (PCOS),metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia,hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes,latent autoimmune diabetes (LAD), and maturity onset diabetes fo theyoung (MODY). In some embodiments, the antibody of the invention is foruse in sensitizing an individual to insulin.

In some embodiments, the invention provides use of an antibody of theinvention in the manufacture of a medicament. In some embodiments, themedicament is for treatment of a metabolic disease or condition selectedfrom the group consisting of: polycystic ovary syndrome (PCOS),metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia,hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes,latent autoimmune diabetes (LAD), and maturity onset diabetes fo theyoung (MODY). In some embodiments, the medicament is for sensitizing anindividual to insulin.

In some embodiments, the invention provides a method of treatingdiabetes in an individual, comprising administering to the individual aneffective amount of an antibody of the invention. In some embodiments,the method further comprises administering to the individual anotheragent to treat diabetes provided that the other agent is not insulin. Insome embodiments, the method further comprises administering to theindividual an agent to treat cardiovascular disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an ELISA measuring binding of anti-FGFR1 antibodies topurified FGFR ECD fragments.

FIG. 1B shows surface plasmon resonance binding constants for R1MAb1 andR1MAb2.

FIG. 1C shows a GAL-Elk1 luciferase assay in rat L6 cells. Cells werecotransfected with an expression vectors for the indicated FGFR isoformtogether with GAL-Elk1, SV40-renilla Luciferase, and Gal-responsivefirefly luciferase reporter. Transfected cells were incubated with mediacontaining increasing concentrations of R1Mab1 or acidic FGF (aFGF:positive control) for 6 hours before luciferase assays. Transcriptionalactivation was assessed by the relative firefly luciferase activitynormalized by renilla luciferase activity and expressed as relativeluciferase unit (RLU).

FIG. 1D shows a GAL-Elk1 luciferase assay in rat L6 cells. Cells werecotransfected with an expression vectors for the indicated FGFR isoformtogether with GAL-Elk1, SV40-renilla Luciferase, and Gal-responsivefirefly luciferase reporter. Transfected cells were incubated with mediacontaining increasing concentrations of R1Mab2 or acidic FGF (aFGF:positive control) for 6 hours before luciferase assays. Transcriptionalactivation was assessed by the relative firefly luciferase activitynormalized by renilla luciferase activity and expressed as relativeluciferase unit (RLU).

FIG. 1E shows an experiment similar to 1C except L6 cells expressed bothFGFR1c and KLB.

FIG. 1F shows an experiment similar to 1C except HEK293 cells were used.

FIG. 1G shows western blot analysis of 3T3-L1 adipocytes treated withindicated protein at 0.5 μg/ml for indicated time.

FIG. 1H shows WAT harvested from lean C57BL/6 mice at indicated timeafter i.p. injection at 1 mpk of R1Mab (+) or control IgG (−), andsubjected to western blot analysis.

FIG. 2A shows blood glucose (left) and body weight (right) of db/db miceafter a single i.p. injection (arrow) of R1Mab1 or control IgG atindicated doses. Significance was observed in glucose (vs control IgG)between day 1-30 for all the groups and in weight (vs control IgG) onday 8 for all the groups, and between day 12-18 for 50 mpk group.N=6˜14, *p<0.05, **p<0.01.

FIG. 2B shows blood glucose at random fed and overnight fasted mice(top) and serum insulin levels after overnight fast, and 30 minutes posti.p. injection of 1 g/kg glucose (bottom). N=6˜14, *p<0.05, **p<0.01.

FIG. 2C shows blood glucose (left) and body weight (right) of Ins2Akitamice after a single i.p. injection of R1Mab1 or control IgG at 3 mpk.N=10. *p<0.01.

FIG. 2D shows food intake (left), blood glucose (center), and bodyweight (right) of db/db mice after a single i.p. injection of R1Mab1 orcontrol IgG at 1 mpk doses. PF: pair-fed to R1MAb-treated group. N=7.#p<0.001 (vs IgG), *p<0.001 (vs PF-IgG).

FIG. 2E shows quantification of insulin positive area in fixedpancreatic sections. The tissues were collected at day 7 post singlei.p. injection of 3 mpk (left) or 1 mpk (right) R1MAb1 and stained forinsulin and glucagon. N=4˜7. **p<0.002. ***p<0.001.

FIG. 3A shows MAPK signaling activation in mouse tissues. Indicatedtissues were harvested at 15 minutes (25 μg/mouse FGF21: top) or 1 hour(1 mpk R1Mab1: bottom) after i.p. injection of lean C57BL/6 mice andsubjected to Western blot analysis. PBS (top) and control IgG (bottom)were used as a negative control.

FIGS. 3B-D shows representative H&E staining of liver (B), hepaticlipids (C), and serum lipids (D). The samples were collected at day 7post single i.p. injection of 1 mpk R1MAb1. Control mice were pair-fedto normalize body weight. N=7, *p<0.05, **p<0.001.

FIG. 3E shows metabolic parameters of ob/ob or transgenic ap2-SREBP1c(srebp) mice injected with 1 mpk Ab. Control groups were pair-fed tonormalize body weight (FIG. S7). Glucose and Insulin for HOMA-IRcalculation was measured on day 3 after 3 hour fast. GTT was conductedwith 1 g/kg i.p. glucose injection on day 4 after overnight fast. Tissueweight was measured on day 5. N=7, *p<0.05, ***p<0.01

FIG. 3F shows metabolic parameters of ap2-SREBP mice subcutaneouslyimplanted with an osmotic pump to infuse FGF21 (12 ng/day). ITT with 1U/kg insulin i.p. injection was conducted on day 4. Serum was collectedon day 6. N=6˜8.

FIG. 4A shows mRNA expression (red: higher expression and blue: lowerexpression) in BAT for genes belonging to indicated KEGG pathway. Thesamples were collected at day 4 post single i.p. injection of 1 mpkR1MAb1 or pair-fed mice injected with control IgG.

FIG. 4B shows mRNA expression in BAT by qPCR. N=6, *p<0.05, **p<0.001.

FIG. 4C shows a luciferase assay in HEK293 cells. The graph shows thatboth FGF21 and R1MAb1 induce transcription of a UAS-driven luciferasereporter gene in HEK293 cells through CREB fused to GAL4 DNA bindingdomain (GAL-CREB) (left two panels), or of a CRE-driven luciferasereporter gene (right two panels), in a dose-dependent manner. Some cellswere also cotransfected to express FGFR1c and KLB (red; top curve ingraphs). The results represent the average of triplicate experiments forthe luciferase activity normalized by renilla activity.

FIG. 4D shows WAT harvested 15 minutes after i.v. injection with 25 μgof FGF21 (+) or PBS (−), and subjected to western blot analysis.

FIG. 4E shows western blot analysis of differentiated primary humanadipocytes treated with FGF21 at 1 μg/ml for 30 minutes.

FIG. 4F shows a model for the signaling pathway through which FGF21 andR1MAb activates the PGC-1alpha program in adipose tissues.

FIG. 5 shows heparin-independent and FGFR1-dependent agonistic activityof R1MAb1. GAL-Elk1 luciferase assay in HEK293 cells. Cells werecotransfected with or without an expression vectors for FGFR1c asindicated, together with GAL-Elk1, SV40-renilla Luciferase, andGal-responsive firefly luciferase reporter. Transfected cells wereincubated in media containing increasing concentrations of R1Mab1 withor without 25 mg/L porcine heparin as indicated for 6 hours beforeluciferase assays. Transcriptional activation was assessed by therelative firefly luciferase activity normalized by renilla luciferaseactivity and expressed as relative luciferase unit (RLU).

FIG. 6A shows food intake (left), body weight (center), and bloodglucose (right) of db/db mice after a single i.p. injection of R1Mab2 orcontrol IgG at 3 mpk doses on day 0. The control mice were pair-fed (PF)to adjust food intake until day 11. At day 11, the food intake ofR1MAb2-treated mice returned to normal, and thus all the mice were fedad libitum after day 11 (AL). N=7˜12. p<0.001.

FIG. 6B shows random fed blood glucose level of mice used in FIG. S2A onday 26.

FIG. 6C shows GTT conducted using the same mice on day 28.

FIG. 6D shows ITT conducted using the same mice on day 37.

FIG. 7A shows blood glucose (left) and body weight (right) of ob/ob miceafter a single i.p. injection of R1Mab1 or control IgG at 1 mpk doses onday 0. The control mice were pair-fed (PF) to R1MAb-treated group. N=7.*p<0.05 (vs PF-IgG).

FIG. 7B shows blood glucose (left) and body weight (right) of HFD-fedC57BL/6 mice after a single i.p. injection of R1Mab1 or control IgG at 1mpk on day 0. N=7˜9. *p<0.05.

FIG. 7C shows GTT conducted HFD-fed mice used in S3B on day 8 post Abinjection. Mice were injected i.p. with 1 g/kg glucose after overnightfast. Mean body weights were 28.6+/−0.6 (R1MAb1) and 32.1+/−0.8 (controlIgG) (p<0.01). N=7.

FIG. 7D shows blood glucose (left) and body weight (right) of Ins2Akitamice on day 5 post single i.p. injection of R1Mab1 or control IgG at 1mpk. The control mice were pair-fed (PF-IgG) to normalize body weight.*p<0.05.

FIG. 8A shows the representative staining of pancreatic islet in db/dbmice analyzed in FIG. 4E. Red: Insulin, Green: Glucagon, Blue: Nuclei.Note that R1MAb did not affect the overall morphology of islets.

FIG. 8B shows the distribution of insulin positive area (%) in eachislet in each animal.

FIG. 9A shows a schematic representation of IgG and the One-Armed (OA)IgG. Blue: Heavy chain, Green: Light Chain. Red asterisk indicatesapproximate position of the residues mutated in the DANA mutant.

FIG. 9B shows a GAL-Elk based luciferase assay in HEK293 cellsexpressing FGFR1c to compare R1MAb2 and the DNA mutant of R1MAb2 (R1MAb2DANA).

FIG. 9C shows blood glucose of db/db mice before (pre) and day 7 posti.p. injection of indicated Ab at 1 mpk. The control mice were pair-fedto normalize body weight.

FIG. 9D shows an ELISA measuring antibody binding to purified FGFR ECDfragments. OA-R1MAb1: OA-version of R1MAb1.

FIG. 9E shows a GAL-Elk based luciferase assay similar to SSB.

FIG. 9F shows western blot analysis of 3T3-L1 adipocytes treated withindicated protein at 0.5 m/ml for indicated time.

FIG. 9G shows blood glucose (left) and body weight (right) of db/db miceafter a single i.p. injection of indicated Ab on day 0 (arrow). N=7.*p<0.05 (control IgG vs 3 mpk R1MAb1), **p<0.0005 (control IgG vs 3 mpkR1MAb1 and control IgG vs 1 mpk R1MAb1).

FIG. 9H shows hepatic and serum lipids from samples were collected onday 7 post Ab injection. N=7. *p<0.05, **p<0.0005 (vs control IgG).

FIG. 10 shows mRNA expression of KLB and FGFR isoforms in liver and WAT.Chow-fed and HFD-fed C57BL/6 mice were 25 weeks old. HFD-fed mice wereon HFD for 21 weeks. *p<0.05 or **p<0.001. N=6.

FIG. 11 shows the requirement for normal adipose function for theactivity of FGF21 and R1MAb. Food intake (Left), blood glucose (center),and body weight (right) of ob/ob or ap2-srebp1c transgenic mice after asingle i.p. injection of R1Mab1 or control IgG at 1 mpk doses on day 0.The same mice described in FIG. 3E. N=7. #p<0.001 (vs IgG), *p<0.001 (vsPF-IgG). The post treatment measurement was done at 1 day postinjection.

FIG. 12A shows a cell-based luciferase assay in HEK293. Cells werecotransfected with an expression vectors for an indicated GAL-fusionproteins, SV40-renilla Luciferase, and GAL-responsive luciferasereporter. Some cell were also cotransfected with expression vector forKLB as indicated. The transfected cells were incubated with mediacontaining conditioned medium from HEK293 cells transfected withexpression vector for FGF21 or control empty vector as indicated. After6 hours of incubation, cells were subjected to luciferase assays.Transcriptional activation was assessed by the relative fireflyluciferase activity normalized by renilla luciferase activity andexpressed as fold induction.

FIG. 12B shows a similar luciferase assay where some cells werecotreated with the following nuclear receptor ligand: 1 MM Wy14643(PPARα), 5 nM GW101516 (PPAR□), 50 nM rosiglitazone (PPARγ), 50 nMT0901317 (LXRα), 5 nM T3 (TRβ), 30 MM CDCA (FXR). Note that FGF21 didnot affected activity of any of the nuclear receptors tested here withor without cognate ligand treatment.

FIG. 12C shows western blot analysis of HEK293 cells treated with FGF21at 0.5 μg/ml for 10 minutes. Some cells were pretreated with aninhibitor for FGFR (100 nM PD173074), mTOR (100 nM rapamycin) MEK1/2 (10μM U0126), PI3K (1 μM wortmannin) as indicated.

FIG. 12D shows downstream genes involved in oxidative metabolism inadipose tissues.

FIG. 13 shows blood glucose (top) and body weight (bottom) of db/db miceafter a single i.p. injection of R1MAb2 (labeled as 182.2), R1MAb3(labeled as 182.5) or control IgG (anti-Her2) at indicated doses. N=6,*p<0.05, **p<0.005, ***p<0.0005. R1MAb3 comprises VH comprising SEQ IDNO: 6 and VH comprising SEQ ID NO: 4.

FIG. 14A shows cumulative food intake, blood glucose, and body weightchange of lean C57BL/6 mice after a single intraperitoneal injection ofR1MAb1 or control IgG at 0.5 mpk. The mice were also implantedsubcutaneously with an osmotic mini-pump on day 0 to continuously infuse(c.i.) recombinant FGF21 at 1.2 mpk/day or vehicle control (PBS). On day3, the mice were overnight fasted to conduct glucose tolerance test(GTT) on day 4 (arrow).

FIG. 14B shows glucose tolerance tests conducted with 2 g/kg glucoseinjected intraperitoneally on day 4 after an overnight fast.

FIG. 14C shows serum analysis of mice shown in FIGS. 2C, S10A and S10B.Serum samples were collected at day 5 after 4 h fast. Data representmean±SEM with n=6 mice per group; *p<0.05, **p<0.01, ***p<0.001 vs. PBS(c.i.)+IgG control, by two-tailed unpaired student's t-test (n.s.=notsignificant).

FIG. 15 shows gene expression analysis using mRNA isolated from livertissue of the mice used in FIGS. 2C and S10. Tissue samples wereisolated on day 5 after 4 h fast. Data represent mean±SEM with n=6 miceper group; *p<0.05, ***p<0.001 vs. PBS (c.i.)+IgG control, by two-tailedunpaired student's t-test (n.s.=not significant).

FIG. 16 shows gene expression analysis using mRNA isolated from brownadipose tissue of the mice used in FIGS. 2C, S10 and S11. Tissue sampleswere isolated on day 5 after 4 h fast. Data represent mean±SEM with n=6mice per group; *p<0.05, ***p<0.001 vs. PBS (c.i.)+IgG control, bytwo-tailed unpaired student's t-test (n.s.=not significant).

FIG. 17A shows ELISA results measuring antibody binding to biotinylatedpeptide fragments.

FIG. 17B shows the amino acid sequences of FGFR1 (amino acids 161-212;SEQ ID NO: 27), the amino acid sequences of peptide #26 (SEQ ID NO: 28)and peptide #28 (SEQ ID NO: 29) along with the amino acids correspondingto peptide #26 from FGFR2 (SEQ ID NO: 30), FGFR3 (SEQ ID NO: 31) andFGFR4 (SEQ ID NO: 32) and the amino acids corresponding to peptide #28from FGFR2 (SEQ ID NO: 33), FGFR3 (SEQ ID NO: 34) and FGFR4 (SEQ ID NO:35). Differences between the peptide #26 and peptide #28 sequences inFGFR1 and the corresponding region of FGFR2-4 are boxed.

FIG. 17C shows ELISA results measuring His-tagged FGFR1 binding to FGF2protein in the presence of various concentrations of R1MAb1 or controlIgG. The data are expressed as % FGFR1-His binding and representmeans±SEM (n=3).

FIG. 17D shows binding of iodinated FGF21 to HEK293 cells stablyexpressing both KLB and FGFR1c in the presence of various concentrationsof non-labeled R1MAb1 or FGF21 (the reaction also contained BSA (10mg/ml) and control IgG (350 mM) to block non-specific binding. The dataare expressed as % bound FGF21 of total radio-labeled FGF21 in thereaction.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

The term “anti-FGFR1 agonist antibody” refers to an antibody that iscapable of binding FGFR1 with sufficient affinity such that the antibodyis useful as a therapeutic agent in activating FGFR1. In one embodiment,the extent of binding of an anti-FGFR1 antibody to an unrelated,non-FGFR1 protein is less than about 10% of the binding of the antibodyto FGFR1 as measured, e.g., by a radioimmunoassay (MA). In certainembodiments, an antibody that binds to FGFR1 has a dissociation constant(Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM(e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹M to10⁻¹³ M).

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ϵ, γ, and μ, respectively.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s).

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-FGFR1 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “Fibroblast Growth Factor Receptor 1 or FGFR1,” as used herein,refers to any native FGFR1 from any vertebrate source, including mammalssuch as primates (e.g. humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessedFGFR1 as well as any form of FGFR1 that results from processing in thecell. The term also encompasses naturally occurring variants of FGFR1,e.g., splice variants or allelic variants. The amino acid sequence ofexemplary human FGFR1b and FGFR2b, respectively, are shown below:

(SEQ ID NO: 1) NTKPNPVAPYWTSPEKMEKKLHAVPAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSVVPSDKGNYTCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKHSGINSSDAEVLTLFNVTEAQSGEYVCKVSNYIGEANQSAWLTVTRPVAKALEERPAVMTS; and (SEQ ID NO: 5)NTKPNPVAPYWTSPEKMEKKLHAVPAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSVVPSDKGNYTCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHEISAWLTVLEALEERPAVMTS.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, decreasing the rate of disease progression,amelioration or palliation of the disease state, and improved prognosis.In some embodiments, antibodies of the invention are used to delaydevelopment of a disease or to slow the progression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

In one aspect, the invention is based, in part, on the discovery ofanti-FGFR1 agonistic antibodies and the therapeutic activity of suchantibodies. Antibodies of the invention are useful, e.g., for thetreatment of metabolic diseases, including diabetes.

A. Exemplary Anti-FGFR1 Antibodies

In one aspect, the invention provides isolated antibodies that bind toFGFR1. In certain embodiments, an anti-FGFR1 antibody binds to FGFR1band/or FGFR1c and agonizes FGFR1 activity.

In one aspect, the invention provides an anti-FGFR1 antibody comprisingat least one, two, three, four, five, or six HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 25; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18 orSEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 22;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23.

In some embodiments, an anti-FGFR1 antibody may be fully human,humanized or non-human. In one embodiment, an anti-FGFR1 antibodycomprises HVRs as in any of the above embodiments, and further comprisesan acceptor human framework, e.g. a human immunoglobulin framework or ahuman consensus framework.

In another aspect, an anti-FGFR1 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 2, 3 or 4 as follows:EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTWISWVPGKGLEWVGEIDPYDGDTYYADSVKGRFTISADTSKNLQMNSLRAEDTAVYYCASSGYGGSDYAMDYWGQ (SEQ ID NO: 2),EVQLVESGGGLVQPGGSLRLSCAASGFTFSNNYIHWVPGKGLEWVADIYPNDGDTDYADSVKGRFTISADTSKNLQMNSLRAEDTAVYYCAREHFDAWVHYYVMDYWGQ (SEQ ID NO: 3), andEVQLVESGGGLVQPGGSLRLSCAASGFTFTSNWISWVPGKGLEWVAEIDPYDGATDYADSVKGRFTISADTSKNLQMNSLRAEDTAVYYCATGTDVMDYWGQ (SEQ ID NO: 4). In certainembodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-FGFR1 antibody comprising that sequenceretains the ability to bind to FGFR1 and to agonize its activity. Incertain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 2, 3 or 4. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti-FGFR1 antibodycomprises the VH sequence in SEQ ID NO: 2, 3 or 4, includingpost-translational modifications of that sequence.

In another aspect, an anti-FGFR1 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 6 as follows:DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKR (SEQ ID NO: 6). Incertain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-FGFR1 antibody comprising that sequenceretains the ability to bind to FGFR1. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 6. In certain embodiments, the substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Optionally, the anti-FGFR1 antibody comprises the VL sequence in SEQ IDNO: 6, including post-translational modifications of that sequence.

In another aspect, an anti-FGFR1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO: 2, 3 or 4 andSEQ ID NO: 6, respectively, including post-translational modificationsof those sequences.

In a further aspect of the invention, an anti-FGFR1 antibody accordingto any of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-FGFR1antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1 antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-FGFR1 antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-ratecan be determined by using a fluorescent quenching technique thatmeasures the increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., 1 Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006).Additional methods include those described, for example, in U.S. Pat.No. 7,189,826 (describing production of monoclonal human IgM antibodiesfrom hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268(2006) (describing human-human hybridomas). Human hybridoma technology(Trioma technology) is also described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for FGFR1 and the other is for any other antigen, e.g.beta-Klotho. In certain embodiments, bispecific antibodies may bind totwo different epitopes of FGFR1. Bispecific antibodies can be preparedas full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to FGFR1 as well asanother, different antigen, e.g. klothoBeta (see, US 2008/0069820, forexample).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “conservative substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFcR expression on hematopoietic cells is summarized in Table 3 on page464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-FGFR1 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-FGFR1 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-FGFR1 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-FGFR1 antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

2. Activity Assays

In one aspect, assays are provided for identifying anti-FGFR1 antibodiesthereof having agonistic activity. For example, biological activity mayinclude the ability to activate signal transduction of particularpathways which can be measured, e.g., by determining levels ofphospho-FRS2a, phospho-MEK, phospho-ERK/MAPK, phospho-STAT3 or using theGAL-Elk1-based luciferase assays described herein (see also, e.g., Wu etal. J. Biol. Chem. 5; 282(40):29069-72 (2007) and Wu et al. PLoS One 18;6(3):e17868 (2011)). Antibodies having such biological activity in vivoand/or in vitro are also provided.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-FGFR1antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinyl sulfone)benzoate) which are commerciallyavailable (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-FGFR1 antibodies provided hereinis useful for detecting the presence of FGFR1 in a biological sample.The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, such as brown adipose tissue, pancreatictissue, liver tissue, white adipose tissue and tumor tissue.

In one embodiment, an anti-FGFR1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of FGFR1 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-FGFR1 antibody as described herein under conditionspermissive for binding of the anti-FGFR1 antibody to FGFR1, anddetecting whether a complex is formed between the anti-FGFR1 antibodyand FGFR1. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-FGFR1 antibody is used to select subjects eligiblefor therapy with an anti-FGFR1 antibody, e.g. where FGFR1 is a biomarkerfor selection of patients.

In certain embodiments, labeled anti-FGFR1 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-FGFR1 antibody as describedherein are prepared by mixing such antibody having the desired degree ofpurity with one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide a glp-1analog, a synthetic amylin, a glucagon receptor antagonist (e.g. ananti-GCGR antibody), or leptin. Such active ingredients are suitablypresent in combination in amounts that are effective for the purposeintended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the agonistic anti-FGFR1 antibodies provided herein may be usedin therapeutic methods.

In one aspect, an agonistic anti-FGFR1 antibody for use as a medicamentis provided. In further aspects, an agonistic anti-FGFR1 antibody foruse in treating a metabolic disease is provided. In certain embodiments,an agonistic anti-FGFR1 antibody for use in a method of treatment isprovided. In certain embodiments, the invention provides an agonisticanti-FGFR1 antibody for use in a method of treating an individual havinga metabolic disease comprising administering to the individual aneffective amount of the anti-FGFR1 antibody. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., a glp-1analog, a synthetic amylin, a glucagon receptor antagonist (e.g. ananti-GCGR antibody), or leptin. An “individual” according to any of theabove embodiments is preferably a human.

In a further aspect, the invention provides for the use of an agonisticanti-FGFR1 antibody in the manufacture or preparation of a medicament.In one embodiment, the medicament is for treatment of a metabolicdisease. In a further embodiment, the medicament is for use in a methodof treating metabolic disease comprising administering to an individualhaving the disease an effective amount of the medicament. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent, e.g.,as described below. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for treating ametabolic disease. In one embodiment, the method comprises administeringto an individual having such metabolic disease an effective amount of anagonistic anti-FGFR1 antibody. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, as described below. An“individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the agonistic anti-FGFR1 antibodies provided herein,e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the agonisticanti-FGFR1 antibodies provided herein and a pharmaceutically acceptablecarrier. In another embodiment, a pharmaceutical formulation comprisesany of the agonistic anti-FGFR1 antibodies provided herein and at leastone additional therapeutic agent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is a glp-1 analog, a synthetic amylin, a glucagon receptorantagonist (e.g. an anti-GCGR antibody), or leptin.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1. Generation and Characterization of Anti-FGFR1 AgonistAntibodies

We generated monoclonal antibodies specific to FGFR1 using phage displaytechnology and His-tagged IgD2-D3 of human FGFR1b and c as antigens.Human phage antibody libraries with synthetic diversities in theselected complementary determining regions (H1, H2, H3, L3), mimickingthe natural diversity of human IgG repertoire were used for panning. TheFab fragments were displayed bivalently on the surface of M13bacteriophage particles (Lee et al., J. Immunol. Methods 284:119-32(2004)). The panning protocol was described previously (Liang et al., J.Mol. Biol. 366:815-29 (2007)). After screening many clones from multiplelibraries, unique and specific phage antibodies that bind to both b andc isoforms of FGFR1 were identified by phage ELISA. To test binding ofthe antibodies to human FGFRs, conventional ELISA protocol was employedusing 2 μg/ml of FGFR1 ECD-human Fc chimeric proteins.

We also measured binding affinities of anti-FGFR1 antibodies to FGFR1were measured by Biacore/SPR using a BIAcore™ T100 instrument asdescribed (Liang et al., supra) with the following modifications. Mouseanti-human Fc antibody was first coated on a BIAcore™ carboxymethylateddextran CM5 chip using direct coupling to free amino groups following aprocedure described by the manufacturer. Antibody was then captured onCM5 biosensor chips to achieve approximately 200 response units (RU).Binding measurements were performed using a running buffer composed of10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% surfactant P20 (HBS-P buffer). A2-fold dilution series of FGFR1 ECD-His protein was injected in a rangeof 1.550 nM in HBS P buffer at a flow rate of 30 μL/minute at 25° C.Association rates (Kon, per mol/s) and dissociation rates (Koff, per s)were calculated using a simple one-one Langmuir binding model (BIAcore™Evaluation Software version 3.2). The equilibrium dissociation constant(Kd, per mol) was calculated as the ratio of Koff/Kon.

Two of the anti-FGFR1 antibodies which were identified as describedabove in independent experiments (designated here as R1MAb1 and R1MAb2)bind to FGFR1b and FGFR1c at a similar affinity, but not to any otherFGFR isoforms (FIGS. 1A and B). Their signaling activity was testedusing a Gal-Elk1 based luciferase assay in rat L6 cells lackingendogenous FGFRs, but transfected to express each FGFR isoform (and KLBas necessary). These antibodies were unexpectedly potent agonists: bothR1MAbs induced luciferase activity in a dose dependent manner, but onlywhen cells express recombinant FGFR1b or FGFR1c, indicating that R1MAbsact as specific agonist for FGFR1 (FIG. 1C). In this assay format,R1MAbs did not appreciably affect activity of basic FGF (bFGF), aclassical FGFR1 ligand (FIG. 1D). However, R1MAbs and FGF21 showed anadditive effect when cells express FGFR1c and KLB (FIG. 1D). F(ab′)2 butnot Fab fragment of R1MAb2 showed FGFR1-dependent agonistic activity,suggesting that the R1MAbs exert their agonistic activity by promotinghomodimerization of FGFR1 (FIG. 1E). Previously, an artificial FGFRdimerizing agonist containing an FGFR1-binding peptide and the c-junleucine zipper domain had been reported. This molecule called C19junalso binds to heparin through the c-jun domain and requires heparin forFGFR1 activation (Ballinger et al., Nature Biotechnol. 17: 1199-1204(1999)). In contrast, heparin did not affect agonistic activity ofR1MAb1 in the luciferase assay (FIG. 5). Heparin-independent agonisticactivity of the R1MAb1 was further confirmed by examiningphosphorylation of ERK and MEK1/2, signaling components downstream ofFGFRs, in cultured murine 3T3-L1 adipocytes (FIG. 1F) or in WAT ofC57BL/6 mice injected intraperitoneally with R1MAb (FIG. 1G).

We performed experiments to map the FGFR1 epitope bound by R1MAb1 andR1MAb2. We synthesized 30 peptides represented portions of the FGFR1sequence with an amino-terminal biotin tag and used them for ELISAbinding assays. These sequences of the peptides were as follows: #1:SSSEEKETDNTKPNPVAPY (SEQ ID NO: 36); #2: PVAPYWTSPEKMEKKLHAV (SEQ ID NO:37); #3: KLHAVPAAKTVKFKCPSSG (SEQ ID NO: 38); #4: CPSSGTPNPTLRWLKNGKE(SEQ ID NO: 39); #5: KNGKEFKPDHRIGGYKVRY (SEQ ID NO: 40); #6:YKVRYATWSIIMDSVVPSD (SEQ ID NO: 41); #7: VVPSDKGNYTCIVENEYGS (SEQ ID NO:42); #8: NEYGSINHTYQLDVVERSP (SEQ ID NO: 43); #9: VERSPHRPILQAGLPANKT(SEQ ID NO: 44); #10: PANKTVALGSNVEFMCKVY (SEQ ID NO: 45); #11:MCKVYSDPQPHIQWLKHIE (SEQ ID NO: 46); #12: LKHIEVNGSKIGPDNLPYV (SEQ IDNO: 47); #13: NLPYVQILKTAGVNTTDKE (SEQ ID NO: 48); #14:TTDKEMEVLHLRNVSFEDA (SEQ ID NO: 49); #15: SFEDAGEYTCLAGNSIGLS (SEQ IDNO: 50); #16: SIGLSHHSAWLTVLEALEE (SEQ ID NO: 51); #17:YWTSPEKMEKKLHAVPAAK (SEQ ID NO: 52); #18: EKMEKKLHAVPAAKTVKFK (SEQ IDNO: 53); #19: PAAKTVKFKCPSSGTPNPT (SEQ ID NO: 54); #20:KFKCPSSGTPNPTLRWLKN (SEQ ID NO: 55); #21: GTPNPTLRWLKNGKEFKPD (SEQ IDNO: 56); #22: TLRWLKNGKEFKPDHRIGG (SEQ ID NO: 57); #23:FKPDHRIGGYKVRYATWSI (SEQ ID NO: 58); #24: HRIGGYKVRYATWSIIMDS (SEQ IDNO: 59); #25: LHAVPAAKTVKFKCPSS (SEQ ID NO: 60); #26: KLHAVPAAKTVKFKCP(SEQ ID NO: 28); #27: AVPAAKTVKFKCPSSG (SEQ ID NO: 61); #28:FKPDHRIGGYKVRY (SEQ ID NO: 29); #29: KPDHRIGGYKVR (SEQ ID NO: 62); #30:GTPNPTLRWLKN (SEQ ID NO: 63). As shown in FIG. 17A, for both R1MAb1 andR1MAb2 the shortest peptides to which they demonstrated significantbinding were peptides #26 and #28.

Example 2. R1MAbs Demonstrate Sustained Anti-Diabetic Activity

We tested whether the anti-FGFR1 antibodies of the invention would haveanti-diabetic activity using mouse models of diabetes. All the mice werepurchased from Jackson Laboratory and maintained in a pathogen-freeanimal facility at 21° C. under standard 12 hr light/12 hr dark cyclewith access to chow (a standard rodent chow (Labdiet 5010, 12.7%calories from fat) or a high fat, high carbohydrate diet (Harlan TekladTD.03584, 58.4% calories from fat) and water ad libitum. db/db mice inC57BLKS/J background were females and other mice were all males. All themice were used for injection around 9-11 weeks old, except ap2-SREBP1cmice were 8 months old (FIG. 3E) or 4 months old (FIG. 3F). Forcontinuous infusion of FGF21 protein, an osmotic pump (Alzet® 2001) wassubcutaneously implanted. Glucose levels were measured using OneTouch®Ultra® glucometer. For hepatic lipid analysis, the lipid was extractedaccording to the Folch method and resuspended in PBS containing 5%Triton X-100. Total cholesterol, triglyceride, β-hydroxybutylate (ThermoDMA) and nonesterified fatty acid (Roche) were determined by usingenzymatic reactions. Serum insulin levels were determined by ELISA(Crystal Chem).

We tested the agonistic activity of the R1MAbs in vitro and in vivo, byinjecting hyperglycemic Leptin-resistant db/db mice with 3, 10, and 50mg/kg (mpk) of R1MAb1. We observed that blood glucose levels werenormalized for over a week at all three doses, and the observedglucose-lowering effect was unexpectedly strong and long-lasting, withglucose levels staying lower than the control mice for over 30 daysafter a single injection (FIG. 2A). This was associated with atransient, but significant decrease in body weight (FIG. 2A). Theglucose lowering effect was likely through an improvement in insulinsensitivity, as serum insulin level was also dramatically decreased byR1MAb1 injection (FIG. 2B). We observed a similar R1MAb-inducedreduction in blood glucose levels with R1MAb2 (FIG. 6), and in threeadditional mouse models with marked insulin resistance, Leptin-deficientob/ob mice, high-fat diet (HFD) fed mice, and Ins2Akita mice (Hong etal., Am. J. Physiol. Endocrinol. Metab. 293:E1687-96 (2007)) (FIGS. 2Cand 7). Pair-feeding experiments showed that R1MAb-induced weight lossin db/db and Ins2Akita mice is due to a reduction in food intake;however, the observed reduction in blood glucose is largely independentof food intake (FIGS. 2D and 6-7). In the pancreas, R1MAb1 injectionincreased insulin positive area per each islet compared with eitherpair-fed or non-pairfed control mice (FIGS. 2E and 8). FGFR1 isexpressed in pancreatic β cells (Hart et al., Nature 408:864-68 (2000))and FGF21 promotes β cell function ex vivo (Wente et al., Diabetes55:2470-78 (2006)); thus activation of FGFR1 in β cells could directlycontribute to the increased pancreatic insulin levels. These datademonstrate that activation of FGFR1 (but not FGFR2 or FGFR3) issufficient to recapitulate anti-diabetic and anti-lipidemic activitiesof recombinant FGF21.

To dissect the importance of the IgG functionalities for R1MAb'sactivities, we utilized two types of modifications. A dual mutation(D265A/N297A; DANA) in the Fc region abolishes binding to FcγRs andrecruitment of immune effector cells by an IgG molecule (Gong et al.,2005). Neither the agonistic nor anti-diabetic activity of R1MAb2 wasaffected by the introduction of the DANA mutations; therefore theeffector function plays no role in anti-diabetic activity of R1MAb2(FIG. 9A-C). However, an engineered one-armed (OA) version of R1Mab1(Atwell et al., J. Mol. Biol. 270:26-35 (1997)), lacking one of the Fabfragments (OA-R1MAb1) (FIGS. 9A and D) showed diminished agonisticactivity (FIGS. 9E and F) and failed to reduce blood glucose, bodyweight, and hepatic and serum lipid levels db/db mice (FIGS. 9G and H),indicating that both the agonistic and anti-diabetic activities ofR1MAbs are dependent on Ab bivalency (although we also test bispecificantibodies with only one anti-FGFR1 arm (e.g., with an anti-beta-Klothoarm) and confirm that they retain the beneficial attributes of thebivalent anti-FGFR1 R1MAbs). These correlations strongly suggest thatR1MAb-induced activation of the signaling pathway downstream of FGFR1likely mediates its anti-diabetic effects.

MAbs have emerged as a powerful therapeutic modality for the treatmentof a number of human diseases. Our demonstration of potentanti-hyperglycemic and lipid-lowering activities of anti-FGFR1 agonisticMAb opens up a novel path towards development of therapeutic MAbstargeting FGFR1 or FGFR1-containing receptor complex for the treatmentof type 2 diabetes and other obesity-related chronic disorders.MAb-based targeting of FGFR1 offers several favorable properties overrecombinant FGF21 therapy. First, by their nature, MAbs providepredictable, modulatable, and far superior pharmacokinetics compared toFGF21 or any other non-antibody therapeutic protein. Indeed, wedemonstrated that a single i.p. injection at 1-3 mpk of R1MAb1 or R1Mab2into db/db mice leads to a remarkably sustained amelioration ofhyperglycemia for over 30 days (FIGS. 2 and 5). Such a long-lastingglycemic effect has never been reported for any of the previouslydescribed anti-diabetic agents.

Example 3. Importance of Adipose Tissues in Diabetic Action of R1MAb andFGF21

Recombinant FGF21 has been suggested to improve insulin sensitivitythrough adipose tissues and the liver (Berglund et al., Endocrinology150:4084-93 (2009); Li et al., FEBS Letters 583:3230-34 (2009)). FGF21injection into mice induced MEK and ERK phosphorylation in fourKLB-expressing tissue types, the liver, white adipose tissue (WAT),brown adipose tissue (BAT), and pancreas, as previously reported (FIG.3A, top) (Kurosu et al., J. Biol. Chem. 282: 26687-95 (2007); Xu et al.,Am. J. Physiol. Endocrinol. Metab. 297(5): E1105-14 (2009)). Incontrast, R1MAb1 injection leads to phosphorylation of the samedownstream effectors in adipose tissues and pancreas, but not in theliver (FIG. 4A, bottom), consistent with very low FGFR1 mRNA expressionin the liver (FIG. 10) (Fon Tacer et al., Mol. Endocrinol. 24(10):2050-64 (2010)). Indeed, a side-by-side comparison of hepatic geneexpression revealed that expression of two previously identifiedFGF21-targeted genes (leptin receptor (LepR) and suppressor of cytokinesignaling 2 (SOCS2); Coskun et al., Endocrinology 149:6018-27 (2008),Inagaki et al., Cell Metabolism 8:77-83 (2008)) were induced by FGF21but not by R1MAb1 (FIG. 15). On the other hand, hepatic expression ofknown insulin regulated genes (acetyl-CoA carboxylase 1 (ACC1), fattyacid synthase (FAS), elongase of long chain fatty acids family 6(Elov16), insulin-like growth factor (IGF)-binding protein (IGFBP)-1,phosphoenolpyruvate carboxykinase (PEPCK)) was similarly altered by bothFGF21 and R1MAb1, suggesting that these genes might be regulatedindirectly via hormonal (e.g. insulin) or metabolic changes. R1MAb1markedly decreased hepatic and serum lipids when injected into db/dbmice at day 7 post single injection, presumably due to lipidrepartitioning effects through adipose tissues (FIG. 3B-D). R1MAb1injection did not induce phosphorylation of MEK or ERK in lung andprostate (FIG. 3A), two cancer-prone tissue types that express FGFR1.These observations together suggest that adipose tissues, but not theliver, are central for the common metabolic activity of R1MAb and FGF21.

To further investigate this point, we used lipoatrophic ap2-srebp1ctransgenic mice, which display severe insulin resistance, leptindeficiency, and hepatomegaly, due to the lack of white adipose tissuesand compromised brown adipose function (FIG. 3E, 11) (Shimomura et al.,Genes Dev. 12:3182-94 (1998); Shimomura et al., Nature 401:73-76(1999)). Consistent with the idea that normal adipose tissue function isrequired for the metabolic activity of R1MAb1, a single i.p. injectionat 1 mpk improved HOMA-IR and glucose tolerance only in the controlob/ob mice but not in ap2-srebp1c mice (FIG. 3E). Food intake wasreduced by R1MAb1 injection in both ob/ob mice and ap2-srebp1ctransgenic mice, when compared to pair-fed mice injected with controlIgG (FIG. 3E). In addition, continuous infusion of recombinant FGF21also failed to improve insulin tolerance in ap2-srebp1c mice, althoughsignificant increase in serum β-hydroxybutyrate (i.e. ketone body) anddecrease in cholesterol were observed (FIG. 3F).

Example 4. PGC1-Alpha Activation in Brown Adipose by R1MAb

FGF21 has recently been suggested to activate the nuclear receptortranscriptional coactivator PGC-1α protein in adipose tissues and theliver to induce expression of the downstream genes associated withoxidative metabolism (Chau et al., Proc. Nat'l. Acad. Sci. USA107:12553-58 (2010); Hondares et al., Cell Metabolism 11:206-12 (2010);Potthoff et al. Proc. Nat'l. Acad. Sci. USA 30; 106(26): 10853-8(2009)). Indeed, when injected into ob/ob mice, R1MAb1 significantlyincreased BAT expression of genes involved in OXPHOS, and fatty acidmetabolism as revealed by DNA microarray analysis followed by a gene-setenrichment analysis (FIG. 4A). R1MAb1 (and FGF21) also increasedexpression of PGC-1a, PGC-1β, and their major targets CIDEA and UCP1 inbrown adipose tissues (measured by qPCR; FIGS. 4B and 16).

Transcription of PGC-1α is regulated through cAMP Response Elements(CREs) in the promoter region and the CREB transcription factor thatbinds to the CREs (Herzig et al., Nature 413:179-83 (2001); Karamitri etal., J. Biol. Chem. 284:20738-52 (2009); Muraoka et al., Am. J. Physiol.Endocrinol. Metab. 296:E1430-39 (2009); Shi et al., J. Biol. Chem.(2005)). In a screen to identify metabolism-related transcriptionfactors that can be activated by FGF21 in HEK293 cells expressing KLB,we found that GAL-CREB fusion protein can be activated by FGF21 (FIG.S8A-B). Subsequently, we found that both FGF21 and R1MAb1 can activateGAL-CREB reporter as well as CRE-luciferase reporter in a dose dependentfashion in HEK293 cells (FIG. 4C). Consistent with the idea that CREBfunctions as a downstream effector, FGF21 increased the phosphorylationof CREB and p90RSK, an upstream CREB kinase regulated by ERK, in mousewhite adipose tissues (FIG. 4D), differentiated human primary adipocyte(FIG. 4E), and HEK293 cells (FIG. 12C). Thus, CREB activation by FGF21and R1MAb likely contributes to induction of PGC-1alpha and a set ofdownstream genes involved in oxidative metabolism in adipose tissues(FIGS. 4F and 12D). In addition to transcriptional regulation suggestedhere, FGF21 has been reported to activate PGC-1α post-translationallyvia activation of AMPK (Chau et al., supra), although we failed toobserve evidence of AMPK activation in vitro or in vivo by R1MAb (datanot shown). Collectively, our results support the role of PGC-1α inadipose tissues in mediating the anti-lipid and anti-diabetic effects ofFGF21 and R1MAb.

Example 5. Testing Bispecific Anti-FGFR1/Anti-Beta-Klotho Antibodies

Another important difference between our R1MAbs and FGF21 is targetreceptor specificity. FGF21 can act on FGFR1c, 2c, and 3c, but itseffects are likely limited to KLB expressing tissues (i.e. liver,adipose, and pancreas) (Fon Tacer et al., supra; Kurosu et al., J. Biol.Chem. 282:26687-95 (2007); Ogawa et al., Proc. Nat'l. Acad. Sci. USA104:7432-37 (2007)). In contrast, the target tissues of our R1MAbs aredetermined by the expression of FGFR1 and tissue distribution of theantibody molecule, but unlikely limited by KLB expression. Indeed, weobserved mild hypophosphatemia in mice treated with R1MAb1, suggestingthe activation of FGF23/Klotho-pathway in the kidney. Accordingly, wegenerated bispecific anti-KLB/FGFR1 bispecific antibodies using phagedisplay or hybridoma technology (BALBc mice immunized with HEK293 cellsexpressing FGFR1c and KLB) to generate separate anti-KLB antibodies andknob-and-hole technology (Merchant et al. Nature Biotechnol. 16(7):677-81 (1998)). We tested the ability of these bispecific antibodies toactivate GAL-Elk1 expression and confirmed that these antibodies dependon the presence of both beta-Klotho and FGFR1 for downstream signalactivation. We also confirmed that one of the antibodies could increasephosphorylation of downstream signaling intermediates, MEK and ERK ½ indifferentiated primary human adipocytes. One of the bispecificantibodies cross reacts with the murine proteins and we used thisantibody to test the in vivo activity of a bispecific antibody. Weobserved that this bispecific antibody reduced blood glucose levelswithout elevating serum FGF23, whereas the corresponding controlanti-FGFR1 (monospecific) antibody reduced blood glucose levels to asimilar extent but significantly elevated serum FGF23 levels.

Next we test the ability of these bispecific antibodies to provide themetabolic benefits of the anti-FGFR1 agonistic antibodies. We generatetransgenic mice expressing human beta-Klotho (the R1MAb1 and R1MAb2 eachrecognize murine FGFR1) and confirm that the anti-KLB/FGFR1 bispecificantibodies described above improve glucose tolerance in mouse models,e.g. high-fat diet fed hKLB transgenic mice. We also generateanti-beta-Klotho antibodies that react with the protein in other modelanimals (e.g. rat, rabbit, cynomologous and rhesus monkeys) andsimilarly test the ability of bispecific antibodies constructed withthese and the anti-FGFR1 antibodies to provide metabolic benefits.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. A nucleic acid molecule encoding ananti-fibroblast growth factor receptor-1 (FGFR1) antibody, wherein theantibody binds to peptide #26 KLHAVPAAKTVKFKCP (SEQ ID NO: 28) orpeptide #28 FKPDHRIGGYKVRY (SEQ ID NO: 29) and wherein the antibody is ahuman, humanized, or chimeric antibody.
 2. A nucleic acid moleculeencoding an anti-FGFR1 antibody, wherein the antibody comprises (a)HVR-H1 comprising the amino acid sequence GFTFTSNWIS (SEQ ID NO: 9), (b)HVR-H2 comprising an amino acid sequence selected from the groupconsisting of AEIDPYDGATDYADSVKG (SEQ ID NO: 14) and EIDPYDGATDYADSVKG(SEQ ID NO: 15), and (c) HVR-H3 comprising an amino acid sequenceselected from the group consisting of TGTDVMDY (SEQ ID NO: 19) andGTDVMDY (SEQ ID NO: 20).
 3. The nucleic acid molecule of claim 2,further comprising (a) HVR-L1 comprising the amino acid sequenceRASQDVSTAVA (SEQ ID NO: 21); (b) HVR-L2 comprising the amino acidsequence SASFLYS (SEQ ID NO: 22); and (c) HVR-L3 comprising the aminoacid sequence QQSYTTPPT (SEQ ID NO: 23).
 4. The nucleic acid molecule ofclaim 1, wherein the antibody is a multispecific antibody.
 5. Thenucleic acid molecule of claim 4, wherein the antibody also binds tobeta-Klotho.
 6. The nucleic acid molecule of claim 2, wherein theantibody is a multispecific antibody.
 7. The nucleic acid molecule ofclaim 6, wherein the antibody also binds to beta-Klotho.
 8. The nucleicacid molecule of claim 3, wherein the antibody is a multispecificantibody.
 9. The nucleic acid molecule of claim 8, wherein the antibodyalso binds to beta-Klotho.
 10. The nucleic acid molecule of claim 1,wherein the antibody is an IgG1 antibody.
 11. The nucleic acid moleculeof claim 2, wherein the antibody is an IgG1 antibody.
 12. The nucleicacid molecule of claim 3, wherein the antibody is an IgG1 antibody. 13.A vector comprising the nucleic acid molecule of any one of claims 1-12.14. A host cell comprising the vector of claim 13.